Critical challenges that impede advances in glioma therapy continue to be the physiological relevance and throughput of preclinical models, and the limited success of single molecule/pathway targeting strategies, likely due to intra-tumoral heterogeneity/rapid adaptive resistance. It has become increasingly apparent that conventional, high throughput 2-D cell culture models diverge substantially from bona fide primary brain tumors. Advanced preclinical models such as tumorsphere and flank patient-derived xenograft or PDX models offer improved retention of some native tumor cell properties, but require weeks to months to establish and still lack native, immune-intact 3-D brain tissue context for glioma cell growth. Moreover, experimental accessibility for these systems is limited, especially for PDX models, where real-time monitoring of tumor growth is precluded, and studies are expensive and time-consuming. As a result, targets and pathways identified in these systems have to date not translated into effective therapeutic strategies. Our most effective current glioma therapy thus continues to be surgical debulking followed by DNA damage delivered by radiation and alkylating chemotherapy. Median overall survival, however, remains at a dismal ~16 months. In this context, the development of effective radiosensitizing agents presents a major opportunity to leverage the current standard-of-care for significantly improved patient outcome. To address this urgent unmet need, the studies proposed here will take advantage of a novel, scalable, immune-competent 3-D organotypic brain slice culture model of glioma we have developed that easily integrates with laboratory models of radiation therapy to create a discovery platform for radiosensitization and resistance targets in glioma. This platform supports both genetic and drug discovery approaches, and we have validated its effective integration with radiation therapy methods at both the target and drug therapy levels. We will use this platform to accomplish a CRISPR-based radiosensitization screen of druggable targets, and to validate their druggability in this model using direct chemical inhibition. Finally, as the platform includes native immune cells as well as resident neurons and glia, we will be able to simultaneously evaluate immune responses and the therapeutic index of novel radiation/compound therapeutic combinations directly at the tissue site of drug action. This new platform combines extensive experience of the Co-PIs in developing elevated-throughput brain- slice assays for drug discovery, and in large-scale genetic screening and both research and clinical radiation oncology. Our goal is to demonstrate and validate this novel approach as an ex vivo brain-slice orthotopic discovery platform to identify radiosensitization targets and to test their druggability for rapid clinical translation towards more effective glioma therapy.